Biomedical subjects

Mira Barda-Saad

Publications and source records attributed to Mira Barda-Saad.

8 recordsLinked to original sources

PubMed2006

Oligomerization of signaling complexes by the multipoint binding of GRB2 to both LAT and SOS1.

Receptor oligomerization is vital for activating intracellular signaling, in part by initiating events that recruit effector and adaptor proteins to sites of active signaling. Whether these distal molecules themselves oligomerize is not well appreciated. In this study, we examined the molecular interactions of the adaptor protein GRB2. In T cells, the SH2 domain of GRB2 binds phosphorylated tyrosines on the adaptor protein LAT and the GRB2 SH3 domains associate with the proline-rich regions of SOS1 and CBL. Using biochemical and biophysical techniques in conjunction with confocal microscopy, we observed that the simultaneous association of GRB2, via its SH2 and SH3 domains, with multivalent ligands led to the oligomerization of these ligands, which affected signaling. These data suggest that multipoint binding of distal adaptor proteins mediates the formation of oligomeric signaling clusters vital for intracellular signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Recruitment and activation of PLCgamma1 in T cells: a new insight into old domains.

Engagement of the T-cell antigen receptor leads to recruitment of phospholipase Cgamma1 (PLCgamma1) to the LAT-nucleated signaling complex and to PLCgamma1 activation in a tyrosine phosphorylation-dependent manner. The mechanism of PLCgamma1 recruitment and the role of PLCgamma1 Src homology (SH) domains in this process remain incompletely understood. Using a combination of biochemical methods and real-time fluorescent imaging, we show here that the N-terminal SH2 domain of PLCgamma1 is necessary but not sufficient for its recruitment. Either the SH3 or C-terminal SH2 domain of PLCgamma1, with the participation of Vav1, c-Cbl and Slp76, are required to stabilize PLCgamma1 recruitment. All three PLCgamma1 SH domains are required for phosphorylation of PLCgamma1 Y783, which is critical for enzyme activation. These novel findings entailed revision of the currently accepted model of PLCgamma1 recruitment and activation in T lymphocytes.

Adaptor Proteins, Signal Transducing↗

PubMed2006

T-cell antigen receptor-induced signaling complexes: internalization via a cholesterol-dependent endocytic pathway.

T-cell antigen receptor engagement causes the rapid assembly of signaling complexes. The adapter protein SLP-76, detected as SLP-yellow fluorescent protein, initially clustered with the TCR and other proteins, then translocated medially on microtubules. As shown by total internal reflection fluorescence microscopy and the inhibition of SLP-76 movement at 16 degrees C, this movement required endocytosis. Immunoelectron microscopy showed SLP-76 staining of smooth pits and tubules. Cholesterol depletion decreased the movement of SLP-76 clusters, as did coexpression of the ubiquitin-interacting motif domain from eps15. These data are consistent with the internalization of SLP-76 via a lipid raft-dependent pathway that requires interaction of the endocytic machinery with ubiquitinylated proteins. The endocytosed SLP-76 clusters contained phosphorylated SLP-76 and phosphorylated LAT. The raft-associated, transmembrane protein LAT likely targets SLP-76 to endocytic vesicles. The endocytosis of active SLP-76 and LAT complexes suggests a possible mechanism for downregulation of signaling complexes induced by TCR activation.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Early phosphorylation kinetics of proteins involved in proximal TCR-mediated signaling pathways.

Activation of T cells via the stimulation of the TCR plays a central role in the adaptive immunological response. Although much is known about TCR-stimulated signaling pathways, there are still gaps in our knowledge about the kinetics and sequence of events during early activation and about the in vivo specificity of kinases involved in these proximal signaling pathways. This information is important not only for understanding the activation of signaling pathways important for T cell function but also for the development of drug targets and computer-based molecular models. In this study, phospho-specific Abs directed toward individual sites on signaling proteins were used to investigate the early phosphorylation kinetics of proteins involved in proximal TCR-induced pathways. These studies indicate that linker for activation of T cells' tyrosines have substantially different phosphorylation kinetics and that Src homology 2 domain-containing leukocyte protein of 76 kDa has rapid, transient phosphorylation kinetics compared to other proteins. In additions, we provide evidence that ZAP-70 is the primary in vivo kinase for LAT tyrosine 191 and that Itk plays a role in the phosphorylation of tyrosine 783 on phospholipase C-gamma1. In total, these studies give new insight into the sequence, kinetics and specificity of early TCR-mediated signaling events that are vital for T cell activation.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Examining multiprotein signaling complexes from all angles.

Dynamic protein-protein interactions are involved in most physiological processes and, in particular, for the formation of multiprotein signaling complexes at transmembrane receptors, adapter proteins and effector molecules. Because the unregulated induction of signaling complexes has substantial clinical relevance, the investigation of these complexes is an active area of research. These studies strive to answer questions about the composition and function of multiprotein signaling complexes, along with the molecular mechanisms of their formation. In this review, the adapter protein, linker for activation of T cells (LAT), will be employed as a model to exemplify how signaling complexes are characterized using a range of techniques. The intensive investigation of LAT highlights how the systematic use of complementary techniques leads to an integrated understanding of the formation, composition and function of multiprotein signaling complexes that occur at receptors, adapter proteins and effector molecules.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Dynamic molecular interactions linking the T cell antigen receptor to the actin cytoskeleton.

T cell receptor (TCR) engagement leads to actin polymerization at the site of T cell contact with antigen-presenting cells. Here we have studied the dynamic activity of proteins involved in regulating actin polymerization in live T cells after activation. Two such adaptor proteins, Nck and the Wiskott-Aldrich syndrome protein (WASp), were recruited to the TCR during initial T cell activation, where they colocalized with the tyrosine kinase Zap70. The recruitment of Nck and WASp depended on TCR-induced tyrosine phosphorylation and the LAT and SLP-76 adaptors. Nck and WASp migrated peripherally and accumulated at an actin-rich circumferential ring. Thus, actin polymerization regulated by the TCR begins at the TCR. Molecules recruited to the TCR regulate actin polymerization and this process drives plasma membrane movement and cellular spreading.

Actins↗

PubMed2003

The mesenchymal stroma negatively regulates B cell lymphopoiesis through the expression of activin A.

The negative control of B cell generation is only partially resolved. We assessed the role of activin A in regulation of B lymphopoiesis in view of its specific inhibitory effects on tumor B lineage cells. Activin A is constitutively expressed in mouse hemopoietic organs and in cultured mesenchymal cell lines. We observed an inverse relationship between activin A titer and B lineage cell production. In the spleen, the red pulp exhibited a relatively higher abundance of the protein as compared with the lymphoid follicles, wherein B cell accumulation occurs. Furthermore, a specific shut off in activin A expression was observed in bone marrow and spleen following in vivo induction of B lymphocyte polyclonal activation. We further substantiated these in vivo observations by in vitro studies of primary bone marrow cultures, in which the expression of functional activin A was found to be diminished prior to the onset of B lymphopoiesis. The reduction in functional activin A is shown to concomitantly occur with spontaneous induction of the expression of activin A specific inhibitors. We therefore propose that the mesenchymal organ stroma expresses activin A that negatively controls B cell lymphopoiesis.

Activins↗

PubMed2002

The mesenchyme expresses T cell receptor mRNAs: relevance to cell growth control.

The mesenchyme plays a crucial regulatory role in organ formation and maintenance. However, comprehensive molecular characterization of these cells is lacking. We found unexpectedly that primary mesenchyme, as well as mesenchymal cell clones, express T cell receptor (TCR)alphabeta mRNAs, lacking the variable region. Immunological and genetic evidence support the expression of a corresponding TCRbeta protein. Additionally, mRNAs encoding TCR complex components including CD3 and zeta chain are present. A relatively higher expression of the mesenchymal TCRbeta mRNA by cultured mesenchymal cell clones correlates with fast growth, whereas poorly expressing cells are slow growers and are contact inhibited. The clones that express relatively higher amount of the TCR mRNA exhibit an increased capacity to form tumors in nude mice. However, the expression of this mRNA in the mesenchyme is not per se leading to tumorigenesis, as demonstrated by primary mesenchyme that does not form tumors in mice while expressing moderate amounts of the TCR transcripts. The expression of mesencymal TCRbeta was confined to the G2/M phases of the cell cycle in the MBA-13 mesenchymal cell line. This cell cycle dependent expression, considered together with the correlation between growth properties and the level of TCR expression by cell clones, implies association of mesenchymal TCR with cell growth control.

Animals↗